1. Field of the Invention
This invention is related to the field of molecular biology, more particularly to methods and compositions involving nucleic acids, and still more particularly to methods and compositions related to methylation analysis using a nicking agent.
2. Description of the Related Art
DNA methylation is important to both prokaryotic and eukaryotic organisms. In prokaryotic organisms, it is involved in DNA replication. Lewin, Genes VII, Oxford University Press, pages 406-8, 2000. In eukaryotic organisms, DNA methylation participates in regulating gene expression, X-chromosome inactivation, genomic imprinting, cell differentiation and tumorigenesis. Rein et al., Nucleic Acids Res. 26: 2255-64, 1998. 5-Methylcytosine is the most abundant methylated nucleotide in eukaryotic cells; while in prokaryotic cells, the most prevalent methylated nucleotides are 5-methylcytosine and N6-methyladenine. Id.
A number of methods have been developed for identifying methylated nucleotides in DNA genomes. These include the use of methylation-sensitive restriction endonucleases and differential base modification by bisulfite, hydrazine or permanganate. Among them, the bisulfite method offers both easy application and high sensitivity. Such high sensitivity is partially a result of PCR amplification of the target nucleic acids after bisulfite treatment. However, because nucleic acid amplification by PCR requires cycles of different temperatures to achieve cycles of denaturation and reannealing, the current bisulfite method requires means for providing cycles of different temperatures. Accordingly, there is a need in the art for a simpler method for determining DNA methylation states.
The present invention fulfills this and related needs. In contrast to previously known techniques, the present invention does not require multiple cycles of different temperatures for amplifying target nucleic acid fragments after being modified.